Aflatoxin Biosynthesis and Type I Polyketide Synthesis

描述(申请人提供)：对环境致癌物质黄曲霉毒素B1的研究将继续进行。这种真菌毒素通过霉菌Aspergitlus fiavus、A.parasiticus和A.Norius污染谷物进入食物供应，这些霉菌产生这种化合物作为正常代谢物。长期摄入会导致肝脏肿瘤，这是亚洲、非洲和中美洲过早死亡的主要原因。在代谢激活形式的毒素和DNA之间的相互作用之间建立了直接的联系，特别是在导致编码蛋白突变的p53基因的“热点”处。P53基因的改变与大约50%的人类癌症有关。对其生物合成的理解提出了生物有机化学中基本感兴趣的问题，并可能提供控制这种环境危害发生的手段。调查有两个主要目标。第一个是了解真菌t型聚酮合成酶(PKSS)--如何完成迭代功能，如何“编程”来控制聚酮链的长度、环化几何构型和氧化状态。这一类的一个特别有趣的例子是黄曲霉毒素生物合成的核心，它是由PKS和催化复合体NORs中的两个专门的酵母样脂肪酸合成酶(FAS)亚单位组成的复合体。NORs的功能将在使用PKS和FASS的表达结构域的“解剖”实验中以及与完整的重组亚单位本身进行检测。一种新开发的识别多结构域蛋白质连接区的算法(UMA)将指导实验单独和成组地检查结构域的功能、物理联系和结构。 第二个目标集中在三个主要的氧化骨架重排步骤，这三个步骤表征了黄曲霉毒素B1从最初形成的蒽醌到最终取代的香豆素的生物合成途径的进展。花色苷A重排为去甲基杂色曲霉毒素，O-甲基杂色曲霉毒素为黄曲霉毒素。基因中断实验已经确定了多达3种介导单个转化的蛋白质，但在每种情况下都确定了一种特定的细胞色素P450。这些反应的机制对它们本身和更广泛的乙酸生素生物合成的背景都是感兴趣的。

DESCRIPTION (provided by applicant): Studies of the potent environmental carcinogen aflatoxin B1 will be continued. This mycotoxin enters the food supply through contamination of grains by the molds Aspergitlus fiavus, A. parasiticus and A. nomius, which produce this compound as a normal metabolite. Chronic ingestion leads to liver tumors that are a major cause of premature death in Asia, Africa and Central America. A direct link has been forged between the interaction of the metabolically activated form of the toxin and DNA, particularly at a "hot spot" in the p53 gene leading to mutation of the encoded protein. Alterations in p53 are associated with ca. 50% of human cancers. An understanding of its biosynthesis presents problems of fundamental interest in bioorganic chemistry and may afford means to control the occurrence of this environmental hazard. The investigation has two major goals. The first seeks understanding of fungal Type t polyketide synthases (PKSs)-how iterative function is accomplished, how "programming" is achieved to control polyketide chain length, cyclization geometry and oxidation state. A particularly interesting example of this class lies at the heart of aflatoxin biosynthesis in a complex of the PKS with two specialized yeast-like fatty acid synthase (FAS) subunits in a catalytic complex, NorS. The function of NorS will be examined both in "dissection" experiments using expressed domains of the PKS and FASs, and with the intact recombinant subunits themselves. A newly developed algorithm (UMA) to identify linker regions in multidomainal proteins will guide the experiments to examine function, physical association and structure of domains individually and in groups. The second goal focuses on the three principal oxidative skeletal rearrangement steps that characterize the progression of the biosynthetic pathway from the initially formed anthraquinone to the final substituted coumarin of aflatoxin B1. These are the rearrangement of averufin to l'-hydroxyversicolorone, versicolorin A to demethylsterigmatocystin, and O-methylsterigmatocystin to aflatoxin. Gene disruption experiments have identified as many as 3 proteins mediating a single transformation, but in each case a particular cytochrome P450 has been identified. The mechanisms of these reactions are of interest in themselves, and in the broader context of acetogenin biosynthesis.